A C35 Carotenoid Biosynthetic Pathway

Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C30 carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C35 backbone. The products of condensation of farnesyldiphosphate and GDP, C35 structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C40 or C30 pathways accepted and converted the C35 substrate, thus creating a C35 carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures

Directed evolution converts subtilisin E into a functional equivalent of thermitase

We used directed evolution to convert Bacillus subtilis subtilisin E into an enzyme functionally equivalent to its thermophilic homolog thermitase from Thermoactinomyces vulgaris. Five generations of random mutagenesis, recombination and screening created subtilisin E 5-3H5, whose half-life at 83°C (3.5 min) and temperature optimum for activity (Topt, 76°C) are identical with those of thermitase. The Topt of the evolved enzyme is 17°C higher and its half-life at 65°C is >200 times that of wild-type subtilisin E. In addition, 5-3H5 is more active towards the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-type at all temperatures from 10 to 90°C. Thermitase differs from subtilisin E at 157 amino acid positions. However, only eight amino acid substitutions were sufficient to convert subtilisin E into an enzyme equally thermostable. The eight substitutions, which include known stabilizing mutations (N218S, N76D) and also several not previously reported, are distributed over the surface of the enzyme. Only two (N218S, N181D) are found in thermitase. Directed evolution provides a powerful tool to unveil mechanisms of thermal adaptation and is an effective and efficient approach to increasing thermostability without compromising enzyme activity

Implications of Rewiring Bacterial Quorum Sensing

Bacteria employ quorum sensing, a form of cell-cell communication, to sense changes in population density and regulate gene expression accordingly. This work investigated the rewiring of one quorum-sensing module, the lux circuit from the marine bacterium Vibrio fischeri. Steady-state experiments demonstrate that rewiring the network architecture of this module can yield graded, threshold, and bistable gene expression as predicted by a mathematical model. The experiments also show that the native lux operon is most consistent with a threshold, as opposed to a bistable, response. Each of the rewired networks yielded functional population sensors at biologically relevant conditions, suggesting that this operon is particularly robust. These findings (i) permit prediction of the behaviors of quorum-sensing operons in bacterial pathogens and (ii) facilitate forward engineering of synthetic gene circuits

Directed enzyme evolution: climbing fitness peaks one amino acid at a time

Directed evolution can generate a remarkable range of new enzyme properties. Alternate substrate specificities and reaction selectivities are readily accessible in enzymes from families that are naturally functionally diverse. Activities on new substrates can be obtained by improving variants with broadened specificities or by step-wise evolution through a sequence of more and more challenging substrates. Evolution of highly specific enzymes has been demonstrated, even with positive selection alone. It is apparent that many solutions exist for any given problem, and there are often many paths that lead uphill, one step at a time

Catalysts on Demand: Selective Oxidations by Laboratory-Evolved Cytochrome P450 BM3

Efficient catalysts for selective oxidation of C-H bonds using atmospheric oxygen are highly desirable to decrease the economic and environmental costs associated with conventional oxidation processes. We have used methods of directed evolution to generate variants of bacterial cytochrome P450 BM3 that catalyze hydroxylation and epoxidation of a wide range of nonnative substrates. This fatty acid hydroxylase was converted to a propane monooxygenase (PMO) capable of hydroxylating propane at rates comparable to that of BM3 on its natural substrates. Variants along the PMO evolutionary lineage showed broadened substrate scope; these became the starting points for evolution of a wide array of enzymes that can hydroxylate and derivatize organic scaffolds. This work demonstrates how a single member of enzyme family is readily converted by evolution into a whole family of catalysts for organic synthesis

Synthetic Gene Circuits: Design with Directed Evolution

Synthetic circuits offer great promise for generating insights into nature's underlying design principles or forward engineering novel biotechnology applications. However, construction of these circuits is not straightforward. Synthetic circuits generally consist of components optimized to function in their natural context, not in the context of the synthetic circuit. Combining mathematical modeling with directed evolution offers one promising means for addressing this problem. Modeling identifies mutational targets and limits the evolutionary search space for directed evolution, which alters circuit performance without the need for detailed biophysical information. This review examines strategies for integrating modeling and directed evolution and discusses the utility and limitations of available methods

Biocatalysts for a biological chemistry: Bringing new chemistry to life

We create enzymes that catalyze reactions not known in living systems. We direct the evolution of new enzymes by starting from the ‘promiscuous’ activities of existing proteins, identifying catalytic activities that may be known to synthetic chemistry but that nature has not (yet) discovered. We have found that heme proteins are a wonderful source of new biochemistry: engineered cytochrome P450s and other heme proteins catalyze a wide range of synthetically useful carbene and nitrene transfer reactions, from alkene cyclopropanation to Si-C bond formation to direct amination of C-H bonds. It’s fascinating to observe how members of nature’s vast catalog of proteins can be evolved—with only a few mutations—to catalyze these reactions with high efficiencies and selectivities, even forming chemical bonds that are unknown in biology. These results demonstrate the ease with which evolution can innovate and enable life to respond to new challenges or opportunities. In the future these fully genetically-encoded catalysts may access vast areas of chemical space that life has not explored. These catalysts already offer an efficient, cost-effective, green biocatalytic alternative to the use of stoichiometric reagents, rare transition metal catalysts, and organic solvents in production of a variety of fine chemicals and pharmaceutical intermediates. Directed Evolution of Cytochrome c for Carbon-Silicon Bond Formation: Bringing Silicon to Life S.B. J. Kan, R. D. Lewis, K. Chen, F. H. Arnold. Science 354, 1048-1051 (2016). Highly Stereoselective Biocatalytic Synthesis of Key Cyclopropane Intermediate to Ticagrelor K. E. Hernandez, H. Renata, R. D. Lewis, S. B. J. Kan, C. Zhang, J. Forte, D. Rozzell, J. A. McIntosh, F. H. Arnold. ACS Catalysis 6, 7810-7813 (2016). Enzyme-Controlled Nitrogen-Atom Transfer Enables Regiodivergent C-H Amination T. K. Hyster, C. C. Farwell, A. R. Buller, J. A. McIntosh, F. H. Arnold. J. Am. Chem. Soc. 136, 15505-15508 (2014) Chemomimetic Biocatalysis: Exploiting the Synthetic Potential of Cofactor-Dependent Enzymes to Create New Catalysts C. K. Prier, F. H. Arnold. J. Am. Chem. Soc. 137, 13992-14006 (2015)

Machine learning-guided directed evolution for protein engineering

Machine learning (ML)-guided directed evolution is a new paradigm for biological design that enables optimization of complex functions. ML methods use data to predict how sequence maps to function without requiring a detailed model of the underlying physics or biological pathways. To demonstrate ML-guided directed evolution, we introduce the steps required to build ML sequence-function models and use them to guide engineering, making recommendations at each stage. This review covers basic concepts relevant to using ML for protein engineering as well as the current literature and applications of this new engineering paradigm. ML methods accelerate directed evolution by learning from information contained in all measured variants and using that information to select sequences that are likely to be improved. We then provide two case studies that demonstrate the ML-guided directed evolution process. We also look to future opportunities where ML will enable discovery of new protein functions and uncover the relationship between protein sequence and function.Comment: Made significant revisions to focus on aspects most relevant to applying machine learning to speed up directed evolutio

Cold Adaptation of a Mesophilic Subtilisin-like Protease by Laboratory Evolution

Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (kcat) at 10 °C 6.6 times and a catalytic efficiency (kcat/KM) 9.6 times that of wild type. Its half-life at 70 °C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in kcat is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77.4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation